The EGF receptor is a classical receptor tyrosine kinase with an extracellular ligand binding domain and an intracellular tyrosine kinase domain. Binding of EGF induces dimerization of the EGF receptor with itself or with other members of the ErbB family of receptor tyrosine kinases with ErbB2 being the preferred dimerization partner. Dimerization of the EGF receptor leads to the stimulation of its intracellular tyrosine kinase activity via the formation of an asymmetric kinase dimer. In this asymmetric dimer, one kinase domain serves as the activator of the other kinase domain. As a result of this mechanism, only one kinase domain can be activated at a time in the receptor dimer. According to the current model, phosphorylation of the receptor then occurs in trans and only takes place in the context of receptor dimers. While appealingly simple, this model cannot explain all aspects of EGFR homodimer and the EGFR/ErbB2 heterodimer function. The EGF receptor has eight different ligands and they are capable of inducing different responses in the same cell line. The molecular basis for this is unknown but it has been hypothesized that the ligands induce different conformations of the EGF receptor. This hypothesis has not been experimentally addressed due to the lack of appropriate tools. Using luciferase fragment complementation imaging, we have documented distinct differences in the response of the EGF receptor to its different ligands, indicating that this method is sensitive to ligand-specific changes in EGF receptor conformation. We have also used radioligand binding studies to document more subtle differences in receptor conformation reflected in differences in the allosteric properties of ligand binding. Therapeutic strategies for targeting ErbB receptors hinge on having a full understanding of how these receptors interact with each other and with their ligands and how they are regulated. The goal of the proposed work is to develop a model of EGFR/ErbB2 activation that is consistent with all extant data and to understand the molecular basis for ligand-specific differences in EGF receptor-mediated signaling. To this end, the specific aims of this grant are to: 1) Develop an accurate model for the activation and phosphorylation in the EGFR/ErbB2 heterodimer; 2) Test the hypothesis that different EGF receptor ligands induce different conformations of the EGF receptor; 3) Quantify ligand-specific differences in the earliest signaling response of the EGF receptor. We will use radioligand binding as well as molecular imaging and traditional kinase assays to test our hypotheses. These studies will provide a more accurate and comprehensive foundation upon which to base research strategies and clinical choices for the development and utilization of ErbB-targeted therapeutics.